N-RAP, a muscle-specific protein concentrated at myotendinous junctions in skeletal muscle and intercalated disks in cardiac muscle, has been implicated in myofibril assembly. To discover more about N-RAP's role in myofibril assembly , we used the yeast two-hybrid system to screen a mouse skeletal muscle cDNA library for proteins capable of binding N-RAP in a eukaryotic cell. From yeast two-hybrid experiments we were able to identify three new N-RAP binding partners: alpha-actinin, filamin-2, and Krp1 (also called sarcosin). In vitro binding assays were used to verify these interactions and to identify the N-RAP domains involved. N-RAP contains an N-terminal LIM domain (N-RAP-LIM), C-terminal actin-binding super repeats homologous to nebulin (N-RAP-SR), and nebulin-related simple repeats in between the two (N-RAP-IB); these three regions of N-RAP were expressed as His-tagged recombinant proteins. We detected significant alpha-actinin binding to N-RAP-IB and N-RAP-LIM, filamin binding to N-RAP-SR, and Krp1 binding to N-RAP-SR and N-RAP-IB. During myofibril assembly in cultured chick cardiomyocytes, N-RAP and filamin appear to colocalize with alpha-actinin in the earliest myofibril precursors found near the cell periphery, as well as in the nascent myofibrils that form as these structures fuse laterally. In contrast, Krp1 is not localized until late in the assembly process, when it appears at the periphery of myofibrils that appear to be fusing laterally. The results suggest that sequential recruitment of N-RAP binding partners may serve an important role during myofibril assembly. Based on biochemical data, immunofluorescence analysis of cultured embryonic chick cardiomyocytes, and the targeting and phenotypic effects of individual GFP-tagged regions of N-RAP, we previously proposed a novel model for the initiation of myofibril assembly in which N-RAP organizes alpha-actinin and actin into the premyofibril I-Z-I complexes. We tested the proposed model by expressing deletion mutants of N-RAP (i.e. constructs containing two of the three regions of N-RAP) in chick cardiomyocytes and observing the effects on alpha-actinin and actin organization into mature sarcomeres. Although individually expressing either the N-RAP-LIM, N-RAP-IB, or N-RAP-SR regions of N-RAP inhibited alpha-actinin assembly into Z-lines, expression of either the N-RAP-LIM-IB fusion or the N-RAP-IB-SR fusion permitted normal alpha-actinin organization. In contrast, the N-RAP-LIM-SR fusion inhibited alpha-actinin organization into Z-lines, indicating that the IB region is critical for Z-line assembly. While permitting normal Z-line assembly, N-RAP-LIM-IB and N-RAP-IB-SR decreased sarcomeric actin staining intensity; however, the effects of N-RAP-LIM-IB on actin assembly were significantly more severe, as estimated both by morphological assessment and by quantitative measurement of actin staining intensity. In addition, N-RAP-LIM-IB was consistently retained in mature Z-lines, while mature Z-lines without significant N-RAP-IB-SR incorporation were often observed. We conclude that the N-RAP super repeats are essential for organizing actin filaments during myofibril assembly in cultured embryonic chick cardiomyocytes, and that they also play an important role in removal of the N-RAP scaffold from the completed myofibrillar structure. The present work strongly supports the N-RAP scaffolding model of premyofibril assembly, for the first time providing a molecular framework for understanding the initial steps used by muscle cells to build the contractile machinery responsible for force and motion. Abnormalities in assembling myofibrils and in linking them to other cellular structures are believed to underlie many diseases of skeletal muscles and the heart. Linkage analysis identified chromosome 10q24-26, a region including the N-RAP gene, as a disease locus for dilated cardiomyopathy (DCM). This past year we described the sequence, genomic structure and expression of human N-RAP, as well as an initial screen to determine whether N-RAP mutations cause cardiomyopathy. Human expressed sequence tag databases were searched with the published 3528 bp mouse N-RAP open reading frame (ORF). Putative cDNA sequences were interrogated by direct sequencing from cardiac and skeletal muscle RNA. We identified two human N-RAP isoforms with ORFs of 5085 bp (isoform C) and 5190 bp (isoform S), encoding products of 193-197 kDa. Genomic database searches localized N-RAP to human chromosome 10q25.3 and matched isoforms C and S to 41 and 42 exons. Only isoform C was detected in human cardiac RNA; in skeletal muscle, approximately 10% is isoform C and approximately 90% is isoform S. We investigated apparent differences between human N-RAP cDNA and mouse sequences. Two mouse N-RAP isoforms with ORFs of 5079 and 5184 bp were identified with ~ 85% similarity to human isoforms; previously published mouse sequences include cloning artifacts truncating the ORF. Murine and human isoforms have similar gene structure, tissue specificity, and size. N-RAP is especially conserved within its LIM domain and super repeat regions. We expressed both N-RAP isoforms and the previously described truncated N-RAP in embryonic chick cardiomyocytes. All constructs targeted to myofibril precursors and the cell periphery, and inhibited myofibril assembly. Several human N-RAP polymorphisms were detected, but none were unique to cardiomyopathy patients. N-RAP is highly conserved and exclusively expressed in cardiac and skeletal muscle. Abnormalities in the N-RAP gene remain excellent candidate causes for cardiac and skeletal myopathies.